The surface membrane proteins of the influenza virus A is composed of three important components, hemagglutinin, neuraminidase, and the M2 channel (Jong, et al. (2006) J. Clin. Virol. 35:2-13), which is a member of the class of proteins termed “viroporins” (Gonzalez & Carrasco (2003) FEBS Lett. 552:28-34). The M2 channel is a homotetrameric protein composed of 97 residues per subunit. Each subunit includes an extracellular N-terminal domain (24 residues), a transmembrane (TM) domain (19 residues), and an intracellular C-terminal domain (54 residues) (Lamb, et al. (1985) Cell 40:627-633; Holsinger & Lamb (1991) Virology 183:32-43; Sugrue & Hay (1991) Virology 180:617-624). The viral M2 protein functions as a proton selective channel, which is activated by low pH environments as found in endosomes (Sakaguchi, et al. (1997) Proc. Natl. Acad. Sci. USA 94:5000-5005). The main functional machinery of the proton-selective M2 channel is believed to lie within the TM helical bundle that exhibits proton conductive activity (Duff & Ashley (1992) Virology 190:485-489).
Amantadine (1-aminoadamantane hydrochloride) and rimantadine (α-methyl-1-adamantane methylamine hydrochloride), an amantadine analogue, are commercial drugs used for the prophylaxis and treatment of influenza A (Oxford, et al. (1980) Pharmacol. Ther. 11:181-262; Dollin, et al. (1982) New Engl. J. Med. 307:580-584) by inhibiting the ion-channel activity of the M2 protein (Pinto, et al. (1992) Cell 517-528; Sugrue & Hay (1991) J. Virol. 180:617-624; Hay (1992) Semin. Virol. 3:21-30). There have been several mechanisms suggested for how the inhibitors interact with the M2 protein. For example, it has been suggested that the inhibitors behave as ‘blockers’ (Hay, et al. (1992) supra; Sansom & Kerr (1993) Prot. Eng. 65-74; Duff, et al. (1994) Virology 202:287-293). In this view, the adamantyl group interacts with Asp24 and Val27 via van der Waals interactions, while the charged amine group hydrogen bonds with Ser31. Alternatively, is has been suggested that amantadine binds to a location deeper in the channel and its ammonium group hydrogen bonds with the His37 side chain (Pinto, et al. (1997) supra; Gandi, et al. (1999) J. Biol. Chem. 274:5474-5482). In this model, binding of the drug blocks proton channel activity by displacing water molecules that are essential for proton conduction. In support of both models, molecular dynamics simulations on the six possible His37 protonation states of M2 in free form and complexed with amantadine and rimantadine indicates that water density in the channel is reduced by the inhibitors (Intharathep, et al. (2008) J. Mol. Graph. Model. 27:342-348).
The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the M2 protein (Klimov, et al. (1995) J. Infect. Dis. 172:1352-1355; Shiraishi, et al. (2003) J. Infect. Dis. 188:57-61). Therefore, there is a need in the art for new drugs to counter the emerging drug resistance. However, one of the major hindrances to the identification and development of antiviral compounds is the lack of suitable models for evaluating compounds with antiviral activity.